One major pathogenic mechanism in obesity is aberrant endocrine signaling, which is exemplified by insulin resistance. Once insulin resistance develops in obesity, hyperlipidemia and hyperglycemia can result, which drives forward pathogenesis. During obesity, macrophages are known to modulate insulin sensitivity through the release of cytokines, among them Interleukin-6 (IL-6). IL-6 is a JAK/STAT ligand, which binds its receptor to activate Janus Kinase, which activates STAT transcription factors. The JAK/STAT pathway is a potential therapeutic target in obesity pathogenesis, but in mammals its use is complicated by nutrient environment specific, ligand specific, and STAT specific effects on insulin sensitivity that can seem contradictory. In Drosophila melanogaster, a state resembling diet-induced obesity results when flies are exposed to a high fat diet (HFD). HFD causes a reduced lifespan, increased triglyceride content, reduced climbing ability, cardiac abnormalities, reduced short term memory, and importantly- insulin resistance. Flies have many analogous physiological systems, including an endocrine fat body functioning as both adipose and liver, a brain with neuroendocrine and pancreas-like cells, muscle, and a partitioned gut including small intestine. The fly JAK/STAT pathway, known as Hop-Stat92E, possesses only a single Janus Kinase (Hopscotch, humans have 4) and a single STAT (Stat92E, humans have 7), meaning that mechanisms of the pathway?s insulin response impact could be more clearly detected through expression profiling relative to mammals. Upd3 (the fly functional ortholog of IL-6) is secreted by macrophages during HFD and Upd3 triggers Hop-Stat92E signaling that results in peripheral insulin resistance through a currently unknown mechanism. Our project seeks to discern mechanisms by which HFD/Upd3 induces peripheral insulin resistance through a simplified JAK/STAT pathway. In Aim 1, we express an overactive form of Hopscotch in each of three tissues with energy homeostasis and endocrine properties: fat body, muscle, and gut. These tissues experience heightened Hop- Stat92E activity during HFD in flies. Upon tissue specific overexpression of Hopscotch using the GAL4-UAS expression system, we will gauge insulin sensitivity through lifespan, triglyceride/glycogen levels, starvation resistance, an oral glucose tolerance test, feeding quantity, qRT-PCR of insulin-like peptide 2 and its receptor, and Western Blot that detects phosphorylation of Akt (Protein Kinase B). In Aim 2, we obtain a gene expression profile using RNA-Sequencing in the Aim 1-identified tissue(s) during ND exposure, HFD exposure, and exposure to each diet but without functional Upd3. Differentially expressed genes that rise in expression during HFD, but that fall when Upd3 is lost, will be prioritized and searched for Stat92E response elements. In Aim 3, Hop-Stat92E regulated genes will be knocked down in the Aim 1 tissue using the GAL4-UAS system, screened for increase tolerance of HFD, and then insulin sensitivity phenotypes from Aim 1 will be monitored. This project will involve student researchers and student coauthorship at the University of Louisiana at Monroe.